STRUCTURAL DETERMINATION OF N-LINKED CARBOHYDRATES BY MATRIX-ASSISTED-LASER-DESORPTION IONIZATION MASS-SPECTROMETRY FOLLOWING ENZYMATIC RELEASE WITHIN SODIUM DODECYL-SULFATE POLYACRYLAMIDE ELECTROPHORESIS GELS- APPLICATION TO SPECIES-SPECIFIC GLYCOSYLATION OF ALPHA(1)-ACID GLYCOPROTEIN/

This paper describes a sensitive method for analysis of N-linked carbo hydrates released enzymatically from within the gel following separati on of glycoproteins (50-100 pmols) by sodium dodecyl sulphate-polyacry lamide gel electrophoresis (SDS-PAGE). The separated bands containing the glycoproteins were cut from the gel, destained, reduced and alkyla ted. N-linked glycans were then released by in-gel incubation with pep tide N-glycosidase-F (PNGase-F) and extracted with water and acetonitr ile. Sialic acid-containing glycans were converted into methyl esters by reaction with methyl iodide, salts and reagents were removed by pas sage through a mixed-bed column of ion-exchange resins and the glycans were examined by matrix-assisted laser desorption/ionization (MALDI)- mass spectrometry. Structural determination of the released glycans wa s performed by exoglycosidase digestion. Following glycan release and extraction, the protein could be digested within the gel with trypsin, and the masses of the tryptic peptides could be compared with those g enerated from a sequence database for protein identification. The meth od is applied to the analysis of N-linked glycans from alpha(1)-acid g lycoprotein from man, cow, sheep and dog. Major species-specific diffe rences in glycosylation were found. Thus, although all four species us ed N-acetyl-neuraminic acid, only cow and sheep additionally used N-gl ycolyl-neuraminic acid. Biantennary glycans were the predominant carbo hydrates in cow, sheep and dog but man produced more triantennary glyc ans and a substantial amount of tetraantennary sugars. Fucosylation wa s only found in glycans from man and cow and both cow and sheep glycan s were found to have beta 1-3- and well as beta 1-4-linked galactose r esidues in the antennae.